The precise partitioning of duplicated chromosomes to daughter cells is essential for the development and survival of all organisms. Defects in segregation lead to aneuploidy, the state where entire chromosomes are gained or lost. Aneuploidy is a hallmark of the majority of tumor cells and has been postulated to be a major factor in the evolution of cancer. Aneuploidy is also the leading cause of spontaneous miscarriages and hereditary birth defects in humans. The goal of this proposal is to elucidate the mechanisms that regulate chromosome segregation and therefore ensure genomic stability. Chromosome segregation requires forces generated by spindle microtubules that are translated into chromosome movement through interactions with kinetochores, the highly conserved structures that assemble onto centromeric chromatin. The simplest eukaryotic kinetochore is in budding yeast where ~38 unique 'core' proteins that exist in 8 subcomplexes assemble into a structure estimated to be >5 MDa. Accurate segregation requires kinetochores to maintain load-bearing attachments to the ends of microtubules that are continually growing and shrinking. Key to proper segregation is the ability of sister kinetochores to biorient and attach to microtubules from opposite poles. Kinetochores also engage a highly conserved signal transduction system called the spindle checkpoint to halt the cell cycle when kinetochore-microtubule interactions are aberrant. To fully elucidate the mechanisms that ensure faithful chromosome segregation, it is critical to understand the diverse functions of kinetochores and their regulation. This proposal will use purified kinetochores and a combination of biochemical, biophysical and structural approaches to address a number of outstanding questions: 1) How does phosphorylation regulate the diverse functions of kinetochores? 2) How do kinetochores regulate the spindle checkpoint? and 3) What is the architecture of a yeast kinetochore? The proposal will use budding yeast for these studies because they are amenable to biochemical, genetic and cytological studies, and the yeast kinetochore is the best characterized to date. Taken together, these studies of kinetochores and their regulation in budding yeast will lead toward an understanding of the fundamental mechanisms of segregation in all eukaryotes. This work will not only elucidate important aspects about the process of segregation, but will aid in the design of better therapeutic interventions in the long-term.